Mechanical Method Of Maintaining Narrow Residence Time Distributions In Continuous Flow Systems

ABSTRACT

Methods of maintaining narrow residence time distributions in continuous flow systems, particularly applicable to virus inactivation such as during a protein purification process. Fluid sample is introduced into an axial flow channel and caused to flow therein in discrete packets or zones to minimize residence time distribution and axial dispersion. Embodiments described herein obviate or minimize the need for using large tanks or reservoirs for performing virus inactivation during a protein purification process; reduce the overall time required for virus inactivation, and/or reduce the overall physical space required to perform the virus inactivation operation during a protein purification process, which in turn reduces the overall footprint for the purification process.

This application claims priority of U.S. Provisional Application Ser.No. 62/504,633 filed May 11, 2017, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND

Large-scale production and the economics around purification oftherapeutic proteins, especially monoclonal antibodies, is anincreasingly important problem for the biopharmaceutical industry.Therapeutic proteins are generally produced in either mammalian cells orbacterial cells which have been engineered to produce the protein ofinterest. However, once produced, the protein of interest needs to beseparated from various impurities such as host cell proteins (HCPs),endotoxins, viruses, DNA etc.

In a typical purification process, the cell culture harvest is subjectedto a clarification step for removal of cell debris. The clarified cellculture harvest containing the protein of interest is then subjected toone or more chromatography steps, which may include an affinitychromatography step or a cation exchange chromatography step. In orderto ensure viral safety of the therapeutic candidate and to comply withregulatory mandates, viral clearance unit operations are implementedinto the purification process. Such steps include Protein A and ionexchange chromatography, filtration and low pH/chemical inactivation.Virus inactivation is typically performed after a chromatography step(e.g. after affinity chromatography or after cation exchangechromatography). In a typical large scale purification process, thechromatographic elution pool containing the protein of interest iscollected in a large tank or reservoir and subjected to a virusinactivation step/process for an extended period of time with mixing,which may take several hours to a day or longer, in order to achievecomplete inactivation of any viruses that may be present in the elutionpool.

In monoclonal antibody (mAb) processing, for example, a sequence ofindependent unit operations is performed in batch mode, where holdingtanks are used to store the material between unit operations andfacilitate any necessary solution adjustments between steps. Typically,the material is collected into one tank where the material is adjustedto achieve the target inactivation conditions. This may be through theaddition of acid to achieve a low pH target level or it may be throughthe addition of detergent in a detergent-based inactivation process.Next, the material is transferred to a second tank where it is held atthe inactivation conditions for a specified incubation time. The purposeof the transfer is to eliminate risk of droplets on the walls of thefirst tank which may not have reached the target inactivation conditionsand could contain virus particles. By transferring the material to adifferent tank, this risk is reduced.

Several virus inactivation techniques are known in the art, includingexposing the protein solution to certain temperatures, pH's, orradiation, and exposure to certain chemical agents such as detergentsand/or salts. One virus inactivation process involves a large holdingtank where material is held at inactivation conditions, such as low pHand/or exposure to detergent, for 60 minutes. This static hold step is abottleneck in moving towards continuous processing.

Virus kill kinetics indicate, however, that the inactivation time couldbe significantly shorter than 60 minutes, which suggests that theprocessing time could be significantly reduced, the static holding tankfor virus inactivation could be eliminated, and the method could be moreamenable to continuous processing and/or flow inactivation.

Recently, there has been a desire to have a continuous process where theunit operations are linked together and manual solution adjustments areminimized. To facilitate this, efforts are being made to develop in-lineprocessing methods to enable in-line virus inactivation as well as otherin-line solution adjustments. A challenge in continuous processing isthe efficient movement of fluid from point A to point B. An examplewould be the plug flow movement of fluid through a length of tubing. Theflow involved in mAb processing typically falls into the laminar flowregime (Reynolds number less than 2100). In this regime, moleculesdisperse due to radial diffusion and as a result, a solute pulse spreadsaxially along the direction of flow. This is known as Taylor dispersion,and is illustrated schematically in FIG. 1. Poiseuille flow for laminarflow leads to a parabolic velocity profile. The leading and trailingends of the pulse begin as sharp interfaces but become parabolic inshape due to the laminar flow of the fluid. The axial spreadingcontinues over time, and the molecules become more disperse over thelength of the tube. The implication of axial dispersion is observed inthe resulting concentration profile obtained for a pulse injection of amarker species at the tube outlet, as seen in FIG. 2. The concentrationprofile reflects a wide distribution of the marker species' residencetime. Such varying residence times may result in uncertainty as towhether all of the fluid has had sufficient residence time in the virusinactivation environment or results in over-sizing the system to securethat no molecules exits sooner than expected. As a consequence ofensuring sufficiently long residence times for virus inactivation, theprotein (product) is exposed to the inactivation conditions forexcessively long residence times which has undesirable consequences suchas potential degradation and aggregation.

In continuous or semi-continuous flow systems, it would be desirable toprovide a method of maintaining narrow residence time distributions.

Provision of a continuous or semi-continuous flow system for biomoleculepurification would be desirable, particularly for protein purification.

SUMMARY

Embodiments disclosed herein provide methods of maintaining narrowresidence time distributions in continuous flow systems, particularlyapplicable to virus inactivation such as during a protein purificationprocess.

Embodiments described herein obviate or minimize the need for usinglarge tanks or reservoirs for performing virus inactivation during aprotein purification process, reduce the overall time required for virusinactivation, and/or reduce the overall physical space required to runthe virus inactivation operation during a protein purification process,which in turn reduces the overall footprint for the purificationprocess. Further, this increases the certainty that all molecules havebeen subjected to a minimum residence time providing some safety factorfor inactivation assurance while minimizing extended holds.

In some embodiments, a method for inactivating one or more viruses thatmay be present in a sample in a purification process is provided, wherethe method comprises maintaining narrow residence distributions incontinuous flow systems by separating the fluid into discrete zones orpackets as it flows in the axial direction of a flow channel, such as atube. The flow channel may function as an incubation chamber. Thisallows for sufficient residence times in the flow channel for allspecies of the fluid, which in turn allows for virus inactivation as thefluid flows in the flow channel and mixes with one or more virusinactivation agents. The flow channel can be made of a variety ofmaterials and shapes, including circular plastic tubing and “SmartFLEXWARE®” macro fluidic flow path assemblies formed by welding twosheets of plastic together in a pattern to create channels, commerciallyavailable from MilliporeSigma (U.S. Pat. Nos. 9,181,941 B2, 9,051,929B2).

In certain embodiments, the incubation chamber, flow channel or tube isconfigured to provide efficient radial mixing and minimal axial mixingthat results in a narrow or reduced residence time distribution, andwherein the volume of the chamber or tube is not subject to variationsdue to pressure and temperature. In some embodiments, the incubationchamber, flow channel or tube is a single use chamber or tube and issterilizable.

In certain embodiments, a method for inactivating one or more virusesthat may be present in a fluid sample containing a target molecule(e.g., an antibody or an Fc region containing protein) is provided,comprising subjecting the fluid sample to a Protein A affinitychromatography process or an ion exchange chromatography process toobtain an eluate; continuously introducing the eluate into an axial flowchannel to mix one or more virus inactivating agents with said eluate inthe flow channel; and causing the eluate to flow in the axial flowchannel in discrete packets for a time sufficient to inactive virus. Incertain embodiments, the chromatography process is carried out in acontinuous mode. The eluate from the affinity chromatography process canbe a real time elution from a column entering the system with all of itsgradients of pH, conductivity, concentration, etc., or can be a pool ofelution then subjected to inactivation after homogenization.

In some embodiments, the in-line incubation chamber, flow channel ortube may be implemented in a process where holding pools or tanks arelocated immediately upstream, downstream, or both, of the chamber,channel or tube. In some embodiments the in-line incubation chamber,flow channel or tube may be implemented in a process wherein thechamber, channel or tube directly connects two unit operations, such asan upstream Protein A chromatography operation and a downstream cationexchange operation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of the flow of a fluid in a channel;

FIG. 2 is a plot of the concentration profile resulting from a pulseinput (a finite volume of a marker species injected into the main streamat a rapid rate so as to create a homogeneous plug of the marker specieswith a concentration profile at each end of the plug approaching a stepchange) of a marker species in a channel in accordance with the priorart;

FIG. 3 is a graph of UV exposure vs. time for various flow channels;

FIG. 4 is a graph of UV exposure vs. volume for various flow channels;

FIG. 5 is a perspective view of apparatus suitable for creating acompressive force on a flow channel to form packets of fluids inaccordance with certain embodiments;

FIG. 6 is a perspective view of a flow channel wound around the mandrelshown in FIG. 5;

FIG. 7 is a perspective view of a flow channel wound around a mandrelplaced in the apparatus of FIG. 5;

FIG. 8 is a graph showing virus inactivation time for various Phi6titers;

FIG. 9 is a graph showing virus inactivation time for various XMuLVretrovirus titers;

FIG. 10 is a graph showing detergent-based virus inactivation kineticsfor Phi 6 titers;

FIG. 11 is a graph of residence time distributions of Phi6 virus incoiled tubing and a conventional flow channel;

FIG. 12 is a schematic diagram of an in-line continuous inactivationprocess in accordance with certain embodiments;

FIG. 13A is a schematic diagram of a windmill design for compressing aflow channel to form packets in accordance with certain embodiments;

FIG. 13B shows top and perspective views of a conveyor design forcompressing a flow channel to form packets in accordance with certainembodiments; and

FIG. 13C is a schematic diagram of a roller design for compressing aflow channel to form packets in accordance with certain embodiments.

DETAILED DESCRIPTION

The term “in-line” or “in-line operation” refers to a process of movinga liquid sample through a tube or some other conduit or flow channelwithout storage in a vessel. The term “virus inactivation” or “viralinactivation” refers to the treatment of a sample containing one or moreviruses in a manner such that the one or more viruses are no longer ableto replicate or are rendered inactive. In methods described herein, theterms “virus” and “viral” may be used interchangeably. Virusinactivation may be achieved by physical means, e.g., heat, ultravioletlight, ultrasonic vibration, or using chemical means, e.g. pH change oraddition of a chemical (e.g., detergent). Virus inactivation istypically a process step which is used during most mammalian proteinpurification processes, especially in case of purification oftherapeutic proteins from mammalian derived expression systems. Inmethods described herein, virus inactivation is performed in a fluidflow channel where the sample is caused to travel in discrete zones orpackets. It is understood that failure to detect one or more viruses ina sample using standard assays known in the art and those describedherein, is indicative of complete inactivation of the one or moreviruses following treatment of the sample with one or more virusinactivating agents.

The term “discrete zone” or “packet” refers to an individually definedvolume separated from adjoining volumes by an intervening barrier.

The term “virus inactivating agent” or “virus inactivation agent” or“virus clearance agent” refers to any physical or chemical means capableof rendering one or more viruses inactive or unable to replicate. Avirus inactivating agent, as used in the methods described herein, mayinclude a solution condition change (e.g. pH, conductivity, temperature,etc.) or the addition of a detergent, a salt, an acid (e.g., aceticacid, with a molarity to achieve a pH of 3.6 or 3.7), a polymer, asolvent, a small molecule, a drug molecule or any other suitable entity,etc., or any combination thereof, which interacts with one or moreviruses in a sample, or a physical means (e.g., exposure to UV light,vibration etc.), such that exposure to the virus inactivating agentrenders one or more viruses inactive or incapable of replicating. In aparticular embodiment, a virus inactivation agent is a pH change, wherethe virus inactivating agent is mixed with a sample containing a targetmolecule (e.g., an eluate from a Protein A bind and elute chromatographystep) in a flow channel where the sample is caused to flow in discretezones or packets.

The term “continuous process” as used herein, includes a process forpurifying a target molecule, which includes two or more process steps(or unit operations), such that the output from one process step flowsdirectly into the next process step in the process, withoutinterruption, and where two or more process steps can be performedconcurrently for at least a portion of their duration. In other words,in the case of a continuous process, it is not necessary to complete aprocess step before the next process step is started, as long as aportion of the sample is always moving through the process steps.

Similarly, a “semi-continuous process” may encompass an operationperformed in a continuous mode for a set period of time with periodicinterruption of one or more unit operations. For example, stopping theloading of feed to allow for the completion of other rate-limiting stepsduring a continuous capture operation.

Conventional processes for protein purification typically involve cellculture methods, e.g., using either mammalian or bacterial cell linesrecombinantly engineered to produce the protein of interest (e.g., amonoclonal antibody) followed by a cell harvest step to remove cell andcell debris from a cell culture broth. The cell harvest step is usuallyfollowed by a capture step, which is typically followed by one or morechromatographic steps, also referred to as polishing steps, whichusually include one or more of cation exchange chromatography and/oranion exchange chromatography and/or hydrophobic interactionchromatography and/or mixed mode chromatography and/or hydroxyapatitechromatography, size exclusion chromatography, depth filtration or useof activated carbon. A virus inactivation step may also be includedafter the capture step. The polishing steps are usually followed byvirus filtration and ultrafiltration/diafiltration, which completes thepurification process.

Biopharmaceutical manufacturing requires the inactivation or removal ofviruses (coming from animal derived components, including mammaliancells) for drug safety and to meet the standards set forth by regulatoryagencies such as the Food and Drug Administration (FDA). Typicalprocesses involve a number of viral clearance steps that cumulativelyprovide the necessary protection.

Some processes involve titration of the solution containing the targetprotein to a low pH in order to cause destruction of any envelopedviruses and viral components. Conventionally, the sample containing thetarget protein is retained at these conditions for an extended period oftime, both because time is needed for virus inactivation but also, andmore importantly, to ensure homogeneous mixing for effective virusinactivation. Therefore, in case of large scale processes, the samplecontaining the target protein is incubated for an extended period oftime at a low pH in order to promote efficient virus inactivation, oftenwith mixing. Two separate tanks are often used, where the first tank isused to adjust the pH and the second tank is used for the actualincubation hold.

The pH conditions are established as a balance between a low pH valuethat is sufficient to cause inactivation and a high enough value toavoid denaturation of the target protein or limit the extent of productdegradation. Additionally, the sample must be exposed for a certainamount of time to cause a significant reduction, usually 2 to 6 LRV (logreduction value) in virus activity values.

Parameters that are considered important for a virus inactivationprocess are pH value, exposure time, the identity of the backgroundsolution conditions (e.g., buffer type, buffer concentration), the mAbconcentration, and temperature, assuming homogeneous mixing is present.In the case of large-scale processes, mixing poses a challenge due tolarge volumes and additional parameters, such as mix rate and masstransfer.

In the case of Fc region containing proteins (e.g. monoclonalantibodies), virus inactivation is usually performed following elutionfrom a bind and elute chromatography process step (e.g., Protein Aaffinity chromatography or cation exchange chromatography) because thepH of the elution pool is closer to the desirable pH for virusinactivation. For example, in processes used in the industry today, theProtein A chromatography elution pool typically has a pH in the 3.5 to4.0 range and the cation exchange bind and elute chromatography elutionpool typically has a pH of about 5.0.

In most processes used in the industry today, the elution poolcontaining the target protein is adjusted to the pH desired for virusinactivation and held there for a certain length of time, thecombination of pH and time having been shown to result in virusinactivation. Longer times are more effective for virus inactivation,especially in case of a large-scale process, however, longer times arealso known to cause protein damage and protein denaturation that canlead to the formation of protein aggregates (immunogenic). Extendedexposure to low pH may result in precipitation and formation ofaggregates, which is undesirable and often requires the use of a depthfilter and/or a sterile filter to remove such precipitates andaggregates.

Methods described herein are able to achieve virus inactivation in acontinuous or semi-continuous manner, which can significantly reduce thetime associated with virus inactivation relative to most conventionalprocesses, and in turn, may reduce the time for the overall purificationprocess.

In some embodiments, the different process steps are connected to beoperated in a continuous or semi-continuous manner. In some embodiments,a virus inactivation method, as described herein, constitutes a processstep in a continuous or semi-continuous purification process, where asample flows continuously from, for example, a Protein A affinitychromatography step or an ion-exchange chromatography step to the virusinactivation step to the next step in the process, which is typically aflow-through purification process step. In-line pH inactivation has beenproposed in prior art (e.g., Klutz S. et al., Continuous viralinactivation at low pH value in antibody manufacturing, ChemicalEngineering and Processing 102(2016) 88-101) but the development of asuitable incubation chamber with a narrow residence time distributionhas meant that these chambers are over-sized and may be more difficultto validate.

In some embodiments, the virus inactivation process step is performedcontinuously or semi-continuously, i.e., the eluate from the previousprocess step, such as the previous bind and elute chromatography step(e.g., Protein A affinity chromatography, FIG. 12) flows continuouslyinto the virus inactivation step, which employs one or more fluidchannels where the eluate is caused to flow in discrete zones orpackets, after which in some embodiments the virus inactivated eluatemay be collected in a storage vessel until the next process step isperformed, or in some embodiments may be fed directly and continuouslyto the next downstream process step. For example, with reference to FIG.12, in certain embodiments Protein A mAb eluate is rapidly brought to auniform low pH using in-line acid addition with precise syringe pumps &static mixers. Robust low pH is maintained over variable protein feedconcentration using 1M acetic acid concentrate. The pH may be verifiedusing sampling and offline sensors. Robust pH control over extendedmultiday operation may be achieved without a complex continuous feedbackcontrol loop and unreliable pH sensors. The inactivation or incubationchamber provides reliable hold time for robust LRV, and a rapidconsistent quench to a pH (e.g., 5-7.5) required for a subsequent step.

In accordance with certain embodiments, narrow residence timedistributions are maintained in continuous or semi-continuous flowsystems. The residence time distributions are sufficiently narrow (andreduced compared to conventional designs) to achieve effective virusinactivation of fluid sample traveling in the system. Suitable narrowresidence time distributions can be quantified based on comparisons toresults obtained from conventional designs. Pulse data (e.g., UVabsorbance peaks) from different designs can be compared usingstatistical quantification metrics. For example, a comparison of theamount of time required for the middle 80% of the fluid to exit a flowchannel can be made. That is, the spread between the 10% and 90% areavalues can be made (where t_(10%) represents the time at which 10% ofthe fluid has exited the channel, and t_(90%) represents the time atwhich 90% of the fluid has exited the channel). This comparison wascarried out for the peaks shown in FIG. 3 and is set forth in Table 1below, using tubing having an ID of ⅛″ and a length of 250 inches toequate to a system volume (hold-up volume) of 50 mL. Other methods ofanalyzing peak characteristics such as those applied by thoseknowledgeable in the art, for example, moment analysis, could also beleveraged.

TABLE 1 Difference t_(10%) t_(90%) between t_(10%) Method (s) (s) andt_(90%) (s) Straight tube 251 1416 1165 Coiled tube 166 403 237 (3/8″rod) The present 231 260 29 flow channel

To compare various designs with different system volumes, these data canalso be presented as a normalized volume (normalized to the systemvolume). This is shown in FIG. 4 and set forth in Table 2 below:

TABLE 2 Difference between (V/ (V/ (V/V_(system))_(10%) and MethodV_(system))_(10%) V_(system))_(90%) (V/V_(system))_(90%) Straight tube0.84 4.72 3.88 Coiled tube 1.02 2.49 1.47 (3/8″ rod) The present 0.961.08 0.12 flow channelIn these analyses examples, the 10-90% spread for embodiments disclosedherein is significantly smaller than the values for the conventionaldesigns, and constitute narrow residence time distributions inaccordance with the embodiments disclosed herein, which are reduced fromthe distributions of conventional designs. In the ideal case for plugflow, this 10-90% spread would go to zero.

In some embodiments, narrow residence time distributions are created andmaintained by using mechanical methods to separate the fluid intodifferent or discrete zones or packets along the axial direction of afluid flow channel in which the sample is traveling. A discrete packetis a packet or zone that is separated from another packet or zone by anintervening barrier; any degree of separation which forms a boundarybetween one volume of the process fluid and an adjacent volume orvolumes of the process fluid is a discrete zone or packet. The barriercan be created by mechanically pinching the walls of the fluid channeltogether (as discussed in greater detail below). The length of thepacket separation (i.e., the compressed region length) is not critical;the packets can be separated by a very small length or a much longerlength, as long as the size is sufficient to maintain the separationbetween packets.

In some embodiments, the different or discrete zones or packets of fluidare achieved by applying compressive forces to the exterior of the fluidflow channel, spaced along the axial length of the channel, while thefluid is flowing in the channel, essentially mimicking a peristalticpump. Separating the fluid into zones or packets minimizes the axialdispersion and mixing of the fluid in the channel. The axial dispersionof residence time will impact the range of residence times a particlemay experience. Longer residence time may be more desirable forinactivating virus particles while at low pH. However, a proteinparticle in the same solution subjected to excessively long residencetime at low pH may degrade, which is undesirable. The extent of theminimization of residence time will depend on the sensitivity of theproduct (e.g., protein) being processed but in any case will bebeneficial the lower it is. Thus the amount of dispersion that can betolerated will depend on the particular application. For virus clearanceapplications, minimizing the axial dispersion will offer significantadvantages in terms of reducing processing time because there will begreater confidence in the virus inactivation within a short window oftime.

In certain embodiments, the formation of discrete packets or zones offluid allows fluid species to be translated axially along the length ofa flow channel such that the profile at the channel inlet matches orsubstantially matches the profile at the channel outlet, addingconsistency and certainty to the residence time of the fluid species inthe channel.

Sample introduced as a pulse at one end of a flow channel will exit theflow channel in a sharp, well-defined peak at a known time. This hasadvantages for in-line continuous virus inactivation applications whereit is necessary to achieve a target minimum residence time for speciesflowing through a system. It also can be implemented in systems wheremobile phase (e.g., buffer) conditions change or where buffer dilutionsoccur. This could take place at multiple places within a typical mAbpurification process. One example is the adjustment that is oftenrequired between a bind-and-elute cation exchange step and aflow-through anion exchange step. The elution pool from the cationexchange step is often at a lower pH and a higher conductivity than thetarget values for the anion exchange step. In order to efficientlyadjust the cation exchange elution pool to the appropriate conditions ina continuous flow system, discrete packets could be formed which wouldallow for an efficient transition to the new conditions. Sharptransitions zones between different buffer types can be provided,thereby minimizing the amount of time and buffer required, and allowingfor in-line buffer dilution or in-line conditioning applications.

Turning now to FIGS. 5-7, there is shown suitable apparatus for applyingcompressive forces to the fluid channel to create the fluid zones orpackets in accordance with embodiments disclosed herein. Many othergeometries and configurations could be designed which would produce thesame effect, such as those illustrated in FIGS. 13A-13C. FIGS. 6 and 7illustrate a mandrel 5 around which a fluid channel 10, such as tubing,can be wrapped to fix the fluid channel 10 in place. One or more rollers15 rotate around the mandrel 5 and apply a constant compressive force tothe outside of the fluid channel 10 as they rotate. The fluid inside ofthe channel 10 is moving axially through the channel as the rollers 15rotate. The pressure from the rollers 15 keeps the fluid separated intomany different zones or packets along the entire length of the fluidchannel 10. The separation into zones or packets minimizes axialdispersion of the fluid and therefore provides flow characteristics thatare desirable for applications involving the efficient movement ofspecies, such as in-line virus inactivation or transitions from onefluid to another, such as buffer transition, or buffer dilution. Theforce necessary to completely close off all of the flow channels acrossone quadrant of the “peristaltic helix” design is a function of the typeand size of the tubing used as the flow channel. For example, the forceneeded to compress 17 revolutions of tubing across one quadrant (eachquadrant separated by 2.23″ of circumference; that is, the compressiveforces are applied every 2.23″, the arc length of tubing betweenrollers) of a helix was 187 lbsf (11 lbsf per revolution of tubing) whenusing reinforced silicone tubing (⅜″ OD, ⅛″ ID). The spacing between therollers dictate the axial length of each packet. Those skilled in theart will appreciate that the foregoing is merely exemplary; otherdesigns could offer other packet sizes.

In some embodiments, a ‘windmill’ design could be used which operates ona radial pattern with changing cross-section to maintain constant fluidvelocity with a constant angular velocity roller driving. One or morerollers may be attached to a rotating hub. As the hub rotates, therollers compress the flow channel, forming fluid packets along the flowchannel. The flow channel can be made of circular plastic tubing or maybe formed from plastic sheets welded together to create channels (e.g.,Smart)FLEXWARE®. FIG. 13A is an example of a mechanical design in whichtubing is arranged in a radial pattern with changing cross-section. Oneor more rollers are used to compress the tubing as the hub rotates. Inthe example shown in FIG. 13A, four rollers are shown on the windmillbut a different number of rollers could be used depending on the desiredfluid packet size.

In some embodiments, a ‘conveyor’ design could be used which involvestwo rolls with a pair of pinch rollers moving across an array of tubing.The pinch rollers traverse along the gap between the two rolls and thencome around again after separating and returning to the other end. Thespacing between the pinch rollers is determined based on the desiredpacket size. FIG. 13B illustrates an example of a mechanical design inwhich tubing is wrapped around two rolls and is pinched by rollerstraversing along the gap between the two rolls.

In some embodiments, rollers could be positioned on the inside of atubing coil such that they compress the tubing against a fixed outerwall. The spacing between the rollers and the tubing size is determinedbased on the desired packet size. FIG. 13C illustrates an example of amechanical compression method in which the rollers are placed on theinside of a tubing coil and the rollers compress the tubing against afixed outer wall.

The flow rate and tubing length may be selected to target a particularresidence time. For virus inactivation applications, the residence timeis chosen as the time sufficient to achieve virus inactivation withinthe flow channel, preferably with some safety factor. For example, wherea 30 minute inactivation time is required, a safety factor of 2 can beemployed, resulting in a target residence time of 60 minutes in the flowchannel. In other embodiments, where virus inactivation takes place inless than 1-2 minutes, a safety factor can be employed and a 4 or 5minute target residence time in the flow channel is used. The minimumresidence time may also depend on regulatory guidance in terms of anacceptable safety factor for virus inactivation.

Suitable nominal residence times include, but are not limited to, 1-2minutes, 2-4 minutes, 4-6 minutes, 6-8 minutes 8-10 minutes, 10-15minutes and 15-30 minutes.

The ability to maintain narrow residence time distributions hasadvantages for virus inactivation processes such as mAb processing whereit is imperative for viruses to spend a minimum amount of time atspecified inactivation conditions such as low pH, exposure todetergents, etc. By maintaining narrow residence time distributions, itis possible to meet the minimum time requirement for virus inactivationwhile also minimizing the exposure of the product (e.g., protein/mAb) tothe harsh inactivation conditions.

In the case of low pH inactivation, for example, virus inactivatingagent such as acid may be added as a side stream into the main feed flowchannel. In certain embodiments, a syringe pump may be used that iscontrolled by a software program to add the desired amount of virusinactivating agent. In some embodiments, a controller for the pump maybe provided, the controller having a processing unit and a storageelement. The processing unit may be a general purpose computing devicesuch as a microprocessor. Alternatively, it may be a specializedprocessing device, such as a programmable logic controller (PLC). Thestorage element may utilize any memory technology, such as RAM, DRAM,ROM, Flash ROM, EEROM, NVRAM, magnetic media, or any other mediumsuitable to hold computer readable data and instructions. Theinstructions may be those necessary to operate the pump. The controllermay also include an input device, such as a touchscreen, keyboard, orother suitable device that allows the operator to input a set ofparameters to be used by the controller. This input device may also bereferred to as a human machine interface or HMI. The controller may haveoutputs adapted to control the pump. These outputs may be analog ordigital in nature, and may provide a binary output (i.e. either on oroff), or may provide a range of possible outputs, such as an analogsignal or a multi-bit digital output. After the agent is added and mixed(e.g., through an in-line static mixer), then it enters the incubationchamber flow channel where it flows through for the target inactivationtime. The agent addition amount depends on the acid type, acid strength,and the buffering capacity of the feed solution. The feed solutionbuffering capacity will depend on many factors including the bufferspecies, buffer concentration, and mAb concentration. An analogousprocess could be used for detergent inactivation instead of low pHinactivation.

In accordance with embodiments disclosed herein, fluid sample introducedinto a fluid channel will exit the fluid channel with minimal peakbroadening. The amount of peak broadening observed when the methodsdisclosed herein are applied is significantly less than whenconventional methods of virus inactivation are employed. FIG. 4illustrates a comparison of peak broadening resulting from the methodsdisclosed herein with conventional methods. Peak broadening with asample injected into a straight tube, a coiled tube (tube wrapped arounda rod with a 3 inch diameter) and a tube where discrete fluid packetsare induced in accordance with embodiments disclosed herein are shown inFIG. 4. All tubes had the same inner diameter and a length whichcorresponds to a 50 mL theoretical hold-up volume. A 0.5 mL sample wasinjected into the tubes initially containing water and after the sampleinjection, water was passed through each tube at 10 mL/min(corresponding to a nominal residence time of 5 minutes). The resultingUV trace was collected at the tube outlet. The volumes were normalizedto the system volume. In the literature, coiled tubes have been shown tooffer advantages over straight tubes due to secondary flow properties(e.g., Dean vortices, U.S. Pat. No. 5,203,002 “Curved channel membranefiltration”). Dean vortices have been implemented specifically forcontinuous virus inactivation (WO2015/135844 A1 “Device and method forcontinuous virus inactivation). The peak obtained using the methodsdisclosed herein is much narrower than that of both the straight tubeand the coiled tube, indicating that the fluid species traveling in theflow channel in discrete packets or zones had a narrower residence timedistribution in the system.

The methods described herein also result in a smaller physical footprintof the process, e.g., by eliminating the need to use a pool tank forvirus inactivation or by minimizing the size of the incubation chambernecessary to inactivate the virus with an appropriate safety factor. Ingeneral, there is a growing demand for more flexible manufacturingprocesses that improve efficiency by reducing the overall physicalfootprint of the process (i.e., floor space). The methods describedherein are able to reduce the overall footprint of a purificationprocess by eliminating large pool tanks that are typically used forvirus inactivation.

EXAMPLES Example 1. Static Low pH Virus Inactivation with BacteriophageVirus

In this representative experiment, the purpose was to evaluate theinactivation kinetics of an enveloped bacteriophage virus (Phi6) at lowpH conditions. The objective was to determine the exposure time requiredfor complete inactivation.

A monoclonal antibody (mAb) was purified by standard Protein Achromatography and prepared at a concentration of 5 mg/mL. The pH wasadjusted from pH 6.3 to pH 3.6 using 8.7 M acetic acid. Human serumalbumin (HSA) (0.25% v/v) was added for Phi6 stability. Followingconfirmation of pH, the virus spike was added to the sample reservoircontaining the mAb and vortexed to ensure a well-mixed system. The Phi6target spike level was 1×10⁷ pfu (plaque forming units)/mL. Within 0.3minutes of the virus addition, a 1 mL sample was removed and transferredto a tube containing a previously determined volume of 2 M Tris Base, pH10 to neutralize (pH 6-8) the sample and quench the inactivation step.The tube was vortexed after the base addition. This process of removingand neutralizing a sample was repeated for each time point. Theseexperiments were carried out at room temperature, 22-25° C. Controlsamples at the initial and final time points were also analyzed. For thecontrol samples, the pH was maintained at the feed pH level (pH 6.3) butthe samples were diluted similarly to the actual samples using thebackground buffer at pH 6.3.

The neutralized samples were assayed for infectivity using the plaqueassay and the results are shown in Table 3. The corresponding Phi6titers are shown in FIG. 8 as the “static” values.

TABLE 3 Time to Complete Inactivation Time (min) Inactivation 1 2 3 4 515 (min) Phi6 Log ≥6.8 ≥6.8 ≥6.8 ≥6.8 ≥6.8 ≥6.8 ≤1 Reduction Values(LRV)

These results indicate that the pH 3.6 conditions rapidly inactivatePhi6, with complete inactivation occurring within 1 minute. Controlsamples at 0 and 15 minutes maintained the original titer level.

Example 2 Static low pH Virus Inactivation with XMuLV Retrovirus

The purpose of this study was to evaluate the virus inactivationkinetics of xenotropic murine leukemia virus (XMuLV, an enveloped viruscommonly used for clearance studies of monoclonal antibody products,under low pH conditions. The objective was to determine the exposuretime required for complete inactivation.

A monoclonal antibody was purified by standard Protein A chromatographyand prepared at a concentration of 18 g/L. The pH was adjusted to atarget level of either pH 3.5, pH 3.7, pH 4.0, or pH 4.2 using 8.7 Macetic acid. Following confirmation of pH, the virus spike was added tothe sample reservoir containing the mAb and vortexed to ensure awell-mixed system. The XMuLV target spike level was 1×10⁷ TCID₅₀/mL(TCID₅₀=Tissue Culture Infection Dose) (4% spike v/v). Within 0.3minutes of the virus addition, a 1 mL sample was removed and transferredto a tube containing a previously determined volume of 2 M Tris Base toneutralize (pH 6-8) the sample and quench the inactivation step. Thetube was vortexed after the base addition. This process of removing andneutralizing a sample was repeated for each time point. Theseexperiments were carried out at room temperature, 22-25° C. Controlsamples at the initial and final time points were also analyzed. For thecontrol samples, the pH was maintained at the feed pH level but thesamples were diluted similarly to the actual samples using thebackground buffer.

The neutralized samples were assayed for infectivity using thecell-based TCID₅₀ infectivity assay using PG4 indicator cells (Bolton G,Cabatingan M, Rubino M, et al. Normal-flow virus filtration:detectionand assessment of the endpoint in bio-processing. Biotechnol ApplBiochem 2005; 42: 133-42). To mitigate cytotoxicity and quench the virusinactivation, samples were diluted 1:50 with ten-fold serial dilutionsin cell culture media and then 100 μl aliquots of each dilution wereadded to a 96-well plate. Following incubation at 37° C. in 5% CO₂ for 7days, infected wells were visually assessed for cytopathic effect (CPE).Titers and LRVs were calculated using standard methods (ICH. Guidance onViral Safety Evaluation of Biotechnology Products Derived From CellLines of Human or Animal Origin. In: Use ICoHoTRfRoPfH, ed. Geneva,Switzerland: ICH, 1998). To determine the LRV at each time point, thetiter of the time point was subtracted from the titer of the closestcontrol time point. The log reduction values are shown in Table 4. Thecorresponding titers are shown in FIG. 9.

TABLE 4 Time to Complete Inactivation Time (min) Inactivation pH 0.3 1 23 4 5 15 30 60 (min) XMuLV 4.0 ≥5.4 ≥5.4 ≥5.4 ≥5.4 ≥5.5 ≥5.5 ≥5.5 ≥5.5≤1 LRV at pH 3.5 XMuLV 3.6 ≥5.5 ≥5.5 ≥5.5 ≥5.5 ≥5.7 ≥5.7 ≥5.7 ≥5.7 ≤1LRV at pH 3.7 XMuLV 1.6 4.7 5.2 5.3 5.2 5.6 ≥5.4 ≥5.4 ≥5.4 ≤15 LRV at pH4.0 XMuLV 1.5 3.3 3.5 3.9 4.0 4.2 4.6 ≥5.7 ≥5.7 ≤30 LRV at pH 4.2

These results indicate that the pH 3.5 and pH 3.7 conditions rapidlyinactivate XMuLV, with complete inactivation occurring within 1 minute.Control samples at 0 and 60 minutes maintained the original titer level.

Example 3 Static Detergent-Based Virus Inactivation with Phi6Bacteriophage Virus

The purpose of this study was to examine detergent-based virusinactivation kinetics. In this representative experiment, two differentconcentrations (0.1% and 1.0% v/v) of a detergent, Triton X-100, wereused to inactivate a bacteriophage virus, Phi6. The objective was todetermine the exposure time required for complete inactivation.

A monoclonal antibody was purified by standard Protein A chromatographyand prepared at a concentration of 15 mg/mL. The virus spike was addedand vortexed to ensure a well-mixed system. The Phi6 target spike levelwas 1×10⁸ pfu/mL. Detergent was added to reach the desiredconcentrations of 0.1% and 1.0% v/v. At various time points, a samplewas removed and quenched by adding it to buffer at a 1:1000 dilutionratio. The tubes were vortexed after the detergent addition. Theseexperiments were carried out at room temperature, 22-25° C. Controlsamples at the initial and final time points were also analyzed. Thecontrol samples were diluted similarly to the actual samples using thebackground buffer.

The neutralized samples were assayed for infectivity using the plaqueassay and the log reduction values are shown in Table 5. Thecorresponding Phi6 titers are shown in FIG. 10.

TABLE 5 Time to Complete Inactivation Time (min) Inactivation 1 2 3 4 515 30 (min) Phi6 LRV ≥5.2 ≥5.2 ≥5.2 ≥5.2 ≥5.2 ≥5.2 ≥5.2 ≤1 at 0.1%Triton X-100 Phi6 LRV ≥6.2 ≥6.2 ≥6.2 ≥6.2 ≥6.2 ≥6.2 ≥6.2 ≤1 at 1.0%Triton X-100

These results indicate that the 0.1% and 1.0% Triton X-100 conditionsrapidly inactivate Phi6, with complete inactivation occurring within 1minute. Control samples at 0 and 30 minutes maintained the originaltiter level.

Example 4 Implementation of Mechanical Method to Create Packets

This representative experiment demonstrates the use of a mechanicalmethod to create packets along a fluid channel.

An apparatus (shown in FIGS. 5-7) was designed to provide compressiveforces at defined intervals along a length of tubing. The tubingdimensions were selected based on the volume required to achieve aparticular residence time at a specified flow rate (i.e., 10 mL/min). Asilicone reinforced tubing with a ⅛″ ID and a ⅜″ OD was used. Thistubing was coiled around a mandrel with a 3″ OD. The mandrel had a helixpath length of 147″. An additional length of tubing connected thisapparatus to a main feed pump and another length of tubing was used onthe outlet side of the incubation chamber.

The tubing coiled around the mandrel was compressed using a set of 4rollers simultaneously. These rollers were supported by an apparatusthat allowed them to be tightened against the tubing, which forced thetubing to be compressed between the mandrel and the rollers. Thismechanically separated the fluid path into packets. The rollers rotatedaround the mandrel at a rotational speed such that the fluid achievedthe target flow rate set by the main feed pump.

Example 5 Residence Time Determination

This representative example provides the method used to determineresidence time distribution.

A peristaltic pump was used to pump a feed solution containing a markerspecies (riboflavin, 0.2 mg/mL) through a tube, an incubation chamber,and into a UV detector. The feed flow rate was 10 mL/min. The incubationchamber utilized the mechanical method described in Example 4 to createpackets of fluid within the system. The incubation chamber consisted of147″ of silicone reinforced tubing (⅛″ ID) inside of the mechanicalapparatus. An additional 30″ of tubing was attached to the inlet andoutlet incubation chamber tubing. These additional tubes had an internaldiameter of ⅛″ and were filled with helical static mixers in order tominimize dispersion. The tubing on the outlet of the incubation chamberconnected the chamber to the UV detector.

The inlet tube was connected to a buffer reservoir. Initially, theentire system was filled with water. With the flow stopped, a fixedvolume (0.5 mL) of marker species (riboflavin) was added into the tubeupstream of the incubation chamber. The flow was set to 10 mL/min andwater was used to push the marker species through the system. The signalfrom the UV detector as a function of time was used to determine themarker species' residence time in the system.

Example 6 Virus Residence Time Distribution

This representative example demonstrates residence time distribution ofa virus through an incubation chamber. The system was set up asdescribed in Example 5 where a mechanical method was implemented tocreate fluid packets within the incubation chamber.

Phi6 virus was used as the marker species. The Phi6 target spike levelwas 1×10⁷ pfu/mL in buffer. The system consisted of a buffer reservoirat the tubing inlet, a length of tubing inside of the incubation chamberwhich was under mechanical compression, and an outlet tube connectingthe incubation chamber to a UV detector. Initially, the system wasfilled with buffer. Then, a 5 mL sample of Phi6 virus was injected intothe system. The flow rate was set to 10 mL/min and buffer was used topush the marker species through the system. Samples were collected atthe tubing outlet and assayed for infectivity using the plaque assay.The results are shown in FIG. 11, where the Phi6 titer is plotted as afunction of normalized volume. The volume was normalized to the systemvolume. These experiments were carried out at room temperature, 22-25°C.

A narrower peak was observed for the current flow channel whichimplemented the mechanical compression method. These results indicatethat the mechanical compression method provided a narrower virusresidence time distribution than the coiled tubing.

Example 7 Virus Inactivation Using a Continuous Flow System

This representative example describes the use of a continuous flowsystem to carry out low pH virus inactivation.

A system consisting of a main feed pump, an acid addition pump, and abase addition pump was used to inactivate virus under continuous flowconditions. A mAb solution was spiked with Phi6 and was connected to themain feed pump inlet. The Phi6 target spike level was 1×10⁸ pfu/mL. Theacid was 1 M acetic acid and the base was 2 M Tris Base solution. Thefeed flow rate was set to 1 mL/min. Acid was added into the main streamat a flow rate of 0.75 mL/min. The fluid then passed through a staticmixer and a length of tubing designed to contain a particular volume(targeting a desired residence time). Different lengths of tubing wereused to contain the required volume for each target average residencetime (1, 2, 3, 4, 5, 15 min) after accounting for the dead volume fromthe valves and pH probe between the acid addition point and thebeginning of the incubation chamber. In all cases, the tubing had aninner diameter of ⅛″. At the outlet of the incubation chamber, thematerial was neutralized by the addition of base into the main line at aflow rate of 0.4 mL/min. The fluid passed through another static mixerand then the neutralized samples were collected at the outlet andassayed for infectivity using the plaque assay. The log reduction valuesare shown in Table 6. These experiments were carried out at roomtemperature, 22-25° C. Control samples were also assayed. For thecontrol samples, the pH was maintained at the feed pH level but thesamples were diluted similarly to the actual samples using thebackground buffer. The corresponding Phi6 titers are shown in FIG. 8 asthe “in-line” values.

TABLE 6 Time to Complete Inactivation Time (min) Inactivation 1 2 3 4 515 (min) Phi6 LRV ≥6.8 ≥6.8 ≥6.8 ≥6.8 ≥6.8 ≥6.8 ≤1

These results indicate that the virus inactivation occurred within 1minute using the in-line continuous inactivation system. Control samplesat 0 and 15 minutes maintained the original titer level.

These data from the in-line system are identical to the data obtained inExample 1 using static low pH inactivation conditions for Phi6 virus,demonstrating equivalence between the two approaches.

1. A method for maintaining a narrow residence time distribution of afluid sample flowing in a fluid channel having an axial length,comprising causing said fluid sample to flow in discrete packets alongsaid axial length within said fluid channel.
 2. The method of claim 1,wherein said fluid sample is caused to flow in discrete packets byapplying compressive force to said fluid channel.
 3. The method of claim1, wherein said fluid sample has a nominal residence time in said fluidchannel of one to two minutes.
 4. The method of claim 1, wherein saidfluid sample has a nominal residence time in said fluid channel of twoto four minutes.
 5. The method of claim 1, wherein said fluid sample hasa nominal residence time in said fluid channel of four to six minutes.6. The method of claim 1, wherein said fluid sample has a nominalresidence time in said fluid channel of six to eight minutes.
 7. Themethod of claim 1, wherein said fluid sample has a nominal residencetime in said fluid channel of eight to ten minutes.
 8. The method ofclaim 1, wherein said fluid sample has a nominal residence time in saidfluid channel of ten to fifteen minutes.
 9. The method of claim 1,wherein said fluid sample has a nominal residence time in said fluidchannel of fifteen to thirty minutes.
 10. A method for inactivating oneor more viruses in a sample containing a target molecule, wherein themethod comprises causing the sample to flow in a flow channel indiscrete packets while continuously exposing the sample to inactivationconditions during a process for purifying said target molecule.
 11. Amethod for inactivating one or more viruses in a fluid sample comprisinga target molecule, comprising subjecting said fluid sample to a ProteinA affinity chromatography process, thereby to obtain an eluate;continuously transferring said eluate to an axial flow channel to mixone or more virus inactivating agents with said eluate; and causing saideluate to flow in said axial channel in discrete packets for a timesufficient to inactive said virus.
 12. A method for inactivating one ormore viruses in a fluid sample comprising a target molecule, comprisingsubjecting said fluid sample to an ion exchange chromatography process,thereby to obtain an eluate; continuously transferring said eluate to anaxial flow channel to mix one or more virus inactivating agents withsaid eluate; and causing said eluate to flow in said axial channel indiscrete packets for a time sufficient to inactive said virus.
 13. Themethod of claim 11, wherein said fluid sample has a nominal residencetime in said fluid channel of one to two minutes.
 14. The method ofclaim 11, wherein said fluid sample has a nominal residence time in saidfluid channel of two to four minutes.
 15. The method of claim 11,wherein said fluid sample has a nominal residence time in said fluidchannel of four to six minutes.
 16. The method of claim 11, wherein saidfluid sample has a nominal residence time in said fluid channel of sixto eight minutes.
 17. The method of claim 11, wherein said fluid samplehas a nominal residence time in said fluid channel of eight to tenminutes.
 18. The method of claim 11, wherein said fluid sample has anominal residence time in said fluid channel of ten to fifteen minutes.19. The method of claim 11, wherein said fluid sample has a nominalresidence time in said fluid channel of fifteen to thirty minutes. 20.The method of claim 11, wherein said eluate is caused to flow indiscrete packets by applying compressive force to said fluid channel.21. The method of claim 11, wherein the target is an antibody or an Fcregion containing protein.
 22. A fluid channel having an axial lengthwith said fluid channel containing a plurality of discrete packets offluid sample containing a target molecule and one or more virusinactivation agents.
 23. The fluid channel of claim 22, wherein saidfluid sample includes a virus.